PROJECT SUMMARY Despite the discovery of ABO(H) blood group antigens and corresponding anti-ABO(H) antibodies over a century ago, anti-ABO(H) antibodies remain one of the most significant immunological barriers to transfusion and transplantation. Moreover, the mechanisms responsible for anti-blood group antibody formation and its regulation remain relatively unknown. Previous studies suggest that exposure to microbes expressing membrane carbohydrate structures resembling ABO(H) antigens may be a driving force in the formation of naturally occurring anti-ABO(H) blood group antibodies. In order to examine the role of blood group expressing microbe exposure in the development of anti-blood group antibody formation, we generated a novel animal model that lacks the mouse blood group B disaccharide (Bdis), generating a recipient that is analogous to human blood group O individuals. Preliminary studies indicate that Bdis negative recipients housed in standard conditions readily produce anti-Bdis antibodies at high titers, while those housed in specific pathogen free (SPF) conditions are unable to produce anti-Bdis antibodies until exposed to Bdis positive microbes in adulthood. Taken together, these findings suggest that microbial exposure may be required for naturally occurring anti-Bdis antibody development. Additional preliminary data demonstrate that exposure to microbes at a younger age results in a greater magnitude of antibody production. However, these high antibody titers fail to persist at constant levels among recipients, which may mirror alterations in sustained colonization of Bdis positive bacteria. These results parallel clinical observations regarding significant differences in anti-ABO(H) antibodies between individuals. Given this variability in antibody levels across age groups and housing conditions of the Bdis negative model, we hypothesize that the timing and duration of microbial exposure impacts the magnitude and persistence of anti-blood group antibody production. To test this hypothesis, we propose the following specific aims: Aim 1. Define the impact of recipient age on the development of anti-Bdis antibodies relevant in anti-ABO(H) hemolytic transfusion reactions. Aim 2. Define the requirement of continued microbial exposure for the persistent production of anti-Bdis antibody relevant to hemolytic transfusion reactions. These studies will provide novel insight into the role blood group expressing microbes play in the production and persistence of anti-ABO(H) antibodies, the most common immunological barrier to transplantation and transfusion therapy.